Project 1. Specification and Synaptogenesis of Excitatory Locomotor Neurons Excitatory interneurons in the ventral spinal cord play important roles in the circuits that control voluntary movements. Very little is known about how these cell types are specified and how they are assembled into functional circuits. In this proposal we will define the genetic program that specifies the V3 class of glutamtergic inteneurons by focusing on the function of two known bHLH transcription factors, Mashl and Ngn3. We will examine the function of V3 interneurons in the locomotor behaviors by genetically ablating or silencing them and assaying motor outputs. We will use a novel genetic assay system that we have developed to look at synaptic connections from V2 and V3 neurons onto other locomotor circuit neurons, including motor neurons and Renshaw cells. We will examine the development of these connections, and ask whether activity plays are role in generating specific patterns of connectivity in the spinal cord with respect to these glutamatergic interneuron synapses. We will also assess the role that the transcription factors Sim1 and Uncx4.1 play in V3 interneuron axon guidance and synapse formation. Finally, we will address the role that Eph-signaling plays in controlling the axon morphology and connectivity of excitatory neurons such as V3 neurons. These studies will begin to map connections between identified locomotor neurons and determine the mechanisms that control their assembly. As such, this research will provide fundamental insights into the development and organization of circuits in the spinal cord that control locomotion, and will lay the ground work for strategies to treat spinal cord injury and degeneration.